In 2010, the Genetics of Coxiella burnetii project (AI 000946) made multiple scientific advances including: 1) Acidified Citrate Cysteine Medium-2 (ACCM-2) supports improved host cell-free growth of Coxiella in liquid and solid media. The historical obligate intracellular nature of Coxiella imposed considerable experimental constraints in defining pathogenic mechanisms. To circumvent this problem, we developed an axenic medium termed Acidified Citrate Cysteine Medium (ACCM) that supports approximately 3 logs of Coxiella growth under microaerobic (2.5% O2) conditions. We have now developed a serum-free version of ACCM termed ACCM-2 that supports improved Coxiella growth in both liquid and solid media. For example, we have increased the colony size on agarose plates by roughly 5-fold to approximately 0.5 mm, greatly aiding clonal isolation by colony picking. Moreover, we now routinely obtain >4 logs of growth in liquid media. ACCM-2 will facilitate biochemical studies and, as described below, aid development of Coxiella genetic systems. 2) Development of new tools for Coxiella genetic manipulation. The lack of methods to genetically manipulate Coxiella significantly impedes study of the organism. We have expanded on our previous success in transforming Coxiella to chloramphenicol resistance and mCherry red fluorescent protein expression using the Himar1 transposon (Tn) system. Resistance to kanamycin has been established as an alternative method of positive selection. Moderate (CBU1169) and strong (CBU0311;outer membrane protein P1) promoters have been defined for driving gene expression in Coxiella. Using Himar1, we have generated mCherry and GFP-expressing Coxiella useful for host-pathogen interaction studies. A shuttle vector was developed by modifying pJB908, originally used in Legionella pneumophila type IV secretion studies, to encode chloramphenicol (pJB-Cat) or kanamycin resistance (pJB-Kan). We currently have clonal pJB-Cat/Kan transformants expressing high levels of selected type IV effector proteins for effector function studies. Moreover, we are expressing dominant/negative proteins in Coxiella using these vectors which will also be critical in complementation studies of forthcoming Coxiella gene knockouts (discussed below). We have developed a system for site-specific, single copy gene knock-ins using Tn7. As observed in other gram-negative bacteria, Tn7 inserts with high frequency into a single intergenic site downstream from Coxiella glmS encoding glucosamine-6-phophosphate synthetase. Applications for Tn7 transgene expression include single copy expression for complementation studies. Finally, suicide plasmids with ColE1/pUC replicons containing a cloned version of a Coxiella gene interrupted with an antibiotic resistance gene are currently being tested to promote allelic exchange. To generate the desired mutation, suicide plasmids require only one cross-over event with subsequent counter-selection to resolve the co-integrant. Indeed, we have established co-integrants in the 3&#8242;and 5&#8242;flanking regions of multiple genes of interest and are now optimizing sacB-mediated sucrose sensitivity as a method of counter-selection to resolve co-integrants and generate gene deletions. Our breakthrough in host cell-free growth of Coxiella has reduced from roughly 3 months to approximately 2 weeks the time required for clonal isolation of transformants. Organisms grown for 7 days in ACCM-2 are electroporated and allowed to recover for 1 day in ACCM-2 without antibiotic. An additional 3 days of growth in ACCM with antibiotic is optimal for transformant colony formation on ACCM-2 agarose. Coxiella plates with high efficiency on ACCM-2 agarose ( 1 to 2 genome equivalents per colony) and the resulting contain clonal populations. Colonies can be expanded by direct transfer into ACCM-2 and resultant liquid cultures stably archived for downstream analyses by using a cryoprotectant and storage at -80 C. 3) Re-sequencing microarrays reveal genetic polymorphisms of clonal phase II isolates likely responsible for LPS phase variation. The high passage phase II isolates in our stock collection are not clonal and contain a small subpopulation of Coxiella still expressing full-length phase I LPS. The resulting mixed genotype complicates identification of indels (insertions/deletions) strictly associated with phase variation. To circumvent this problem, we used our micromanipulation cloning procedure to isolate clonal phase II populations of high passage Nine Mile, Australia and California isolates. By hybridizing their genomic DNA to a high-density microarray that contains probe sets encompassing the entire genome of the Nine Mile phase I isolate, we identified common indels in phase II organisms that may account for defective LPS biosynthesis. 4) Subunit vaccine candidates can be identified by high-throughput T cell antigen screens employing proteins produced by in vitro transcription/translation. Using a Coxiella protein microarray spotted with proteins produced by in vitro transcription and translation, we previously identified immunodominant antigens recognized by antibody in the context of human Coxiella infection or vaccination. While the primary goal of this study was to identify Coxiella proteins with serodiagnostic potential, some of the identified antigens may be candidates for a Q fever subunit vaccine based on recombinant protein. To this end, and in collaboration Dr. Wendy Brown at Washington State University, we tested whether IVTT-produced protein coupled to latex beads could be recognized by memory CD4+ T cells. The technique was optimized using Dr. Browns well-developed Anaplasmosis cattle model. Fifty bioinformatically-selected Anaplasma marginale proteins, along with some known Anaplasma T cell antigens, were expressed by IVTT and bead-affinity purified using antibodies to His and FLAG epitope tags. IVTT-expressed bead-bound antigens were processed and presented by antigen presenting cells to T cells from immunized animals and evaluated for immunogenicity in proliferation assays. Known T-cell antigens consistently stimulated T cell responses. Importantly, the study also identified 5 new T-cell antigens among the 50 screened proteins. This method can now be adapted to quickly screen the Coxiella proteome for antigens that react with T-cells derived from vaccinated/infected animals. Collectively, these efforts directly address NIAIDs biodefense goal of developing new countermeasures against Q fever such as rationally designed subunit vaccines and sero-diagnostic reagents.